Glycosaminoglycan-synthetic polymer conjugates

ABSTRACT

Pharmaceutically acceptable, nonimmunogenic compositions are formed by covalently binding glycosaminoglycans or derivatives thereof, to hydrophilic synthetic polymers via specific types of chemical bonds to provide biocompatible conjugates. Useful glycosaminoglycans include hyaluronic acid, the chondroitin sulfates, keratan sulfate, chitin and heparin, each of which is chemically derivatized to react with a hydrophilic synthetic polymer. The conjugate comprising a glycosaminoglycan covalently bound to a hydrophilic synthetic polymer may be further bound to collagen to form a three component conjugate having different properties. The hydrophilic synthetic polymer may be polyethylene glycol and derivatives thereof having an average molecular weight over a range of from about 100 to about 100,000. The compositions may include other components such as fluid, pharmaceutically acceptable carriers to form injectable formulations, and/or biologically active proteins such as growth factors or cytokines. The conjugates of the invention generally contain large amounts of water when formed. The conjugates can be dehydrated to form a relatively solid implant for use in hard tissue augmentation. The dehydrated, solid implant can further be ground into particles which can be suspended in a non-aqueous fluid and injected into a living being (preferably human) for soft tissue augmentation. Once in place, the solid implants or particles rehydrate and expand in size approximately three- to five-fold.

CROSS-REFERENCES

This application is a Divisional of copending U.S. application Ser. No.08/146,843, filed Nov. 3, 1993, which is a continuation-in-part of U.S.application Ser. No. 07/907,518, filed Jul. 2, 1992, and now U.S. Pat.No. 5,324,775, issued Jun. 28, 1994, which is a continuation-in-part ofU.S. application Ser. No. 07/433,441, filed Nov. 14, 1989, and now U.S.Pat. No. 5,162,430, issued Nov. 10, 1992, which is acontinuation-in-part of U.S. application Ser. No. 07/274,071, filed Nov.21, 1988, subsequently abandoned, which applications and issued patentsare incorporated herein by reference in full and to which we claimpriority under 35 USC §120.

FIELD OF THE INVENTION

This invention relates to biocompatible conjugates and, specifically, topharmaceutically acceptable, nonimmunogenic compositions comprising oneor more glycosaminoglycans, or derivatives thereof, conjugated to asynthetic hydrophilic polymer such as polyethylene glycol (PEG), whichis optionally conjugated to collagen as well.

BACKGROUND OF THE INVENTION

Daniels et al, U.S. Pat. No. 3,949,073, disclosed the preparation ofsoluble collagen by dissolving tissue in aqueous acid, followed byenzymatic digestion. The resulting atelopeptide collagen is soluble, andsubstantially less immunogenic than unmodified collagen. It may beinjected into suitable locations of a subject with a fibril-formationpromoter (described as a polymerization promoter in the patent) to formfibrous collagen implants in situ, for augmenting hard or soft tissue.This material is now commercially available from Collagen Corporation(Palo Alto, Calif.) under the trademark Zyderm® Collagen Implant.

Luck et al, U.S. Pat. No. 4,488,911, disclosed a method for preparingcollagen in solution (CIS), wherein native collagen is extracted fromanimal tissue in dilute aqueous acid, followed by digestion with anenzyme such as pepsin, trypsin, or Pronase®, a trademark of AmericanHoechst Corporation, Somerville, N.J. The enzymatic digestion removesthe telopeptide portions of the collagen molecules, providing"atelopeptide" collagen in solution. The atelopeptide CIS so produced issubstantially nonimmunogenic, and is also substantially non-crosslinkeddue to loss of the primary crosslinking regions. The CIS may then beprecipitated by dialysis in a moderate shear environment to producecollagen fibers which resemble native collagen fibers. The precipitated,reconstituted fibers may additionally be crosslinked using a chemicalagent (for example, aldehydes such as formaldehyde and glutaraldehyde),heat, or radiation. The resulting products are suitable for use inmedical implants due to their biocompatability and reducedimmunogenicity.

Wallace et al, U.S. Pat. No. 4,424,208, disclosed an improved collagenformulation suitable for use in soft tissue augmentation. Wallace'sformulation comprises reconstituted fibrillar atelopeptide collagen (forexample, Zyderm® Collagen) in combination with particulate, crosslinkedatelopeptide collagen dispersed in an aqueous medium. The addition ofparticulate crosslinked collagen improves the implant's persistence, orability to resist shrinkage following implantation.

Smestad et al, U.S. Pat No. 4,582,640, disclosed a glutaraldehydecrosslinked atelopeptide CIS preparation (GAX) suitable for use inmedical implants. The collagen is crosslinked under conditions favoringintrafiber bonding rather than interfiber bonding, and provides aproduct with higher persistence than non-crosslinked atelopeptidecollagen. Said product is commercially available from CollagenCorporation under the trademark Zyplast® Collagen Implant.

Nguyen et al, U.S. Pat. No. 4,642,117, disclosed a method for reducingthe viscosity of atelopeptide CIS by mechanical shearing. Reconstitutedcollagen fibers are passed through a fine-mesh screen until viscosity isreduced to a practical level for injection.

Nathan et al, U.S. Pat. No. 4,563,350, disclosed osteoinductive bonerepair compositions comprising an osteoinductive factor, at least 5%nonreconstituted (afibrillar) collagen, and the remainder reconstitutedcollagen and/or mineral powder (e.g., hydroxyapatite). CIS may be usedfor the nonreconstituted collagen, and Zyderm® Collagen Implant (ZCI) ispreferred for the reconstituted collagen component. The material isimplanted in bone defects or fractures to speed ingrowth of osteoclastsand promote new bone growth.

Chu, U.S. Pat. No. 4,557,764, disclosed a "second nucleation" collagenprecipitate which exhibits a desirable malleability and putty-likeconsistency. Collagen is provided in solution (e.g., at 2-4 mg/mL), anda "first nucleation product" is precipitated by rapid titration andcentrifugation. The remaining supernatant (containing the bulk of theoriginal collagen) is then decanted and allowed to stand overnight. Theprecipitated second nucleation product is collected by centrifugation.

Chu, U.S. Pat. No. 4,689,399, disclosed a collagen membrane preparation,which is prepared by compressing and drying a collagen gel. Theresulting product has high tensile strength.

Silver et al., U.S. Pat. No. 4,703,108, disclosed the preparation of asponge prepared by crosslinking insoluble collagen using dehydrothermalmeans or by using cyanamide. Berg et al., U.S. Pat. No. 4,837,285,disclosed the preparation of collagen in bead form for soft tissueaugmentation. Brodsky et al., U.S. Pat. No. 4,971,954, have disclosed amethod of crosslinking collagen using ribose or other reducing sugars.

Miyata et al., Japanese patent application 4-227265, published Aug. 17,1992, discloses a composition comprised of atelopeptide collagen linkedto a polyepoxy compound. The composition is injected into the body toobtain sustained skin-lifting effects.

J. A. M. Ramshaw et al, Anal Biochem (1984) 141:361-65, and PCTapplication WO87/04078, disclosed the precipitation of bovine collagen(types I, II, and III) from aqueous PEG solutions, where there is nobinding between collagen and PEG.

Werner, U.S. Pat. No. 4,357,274, disclosed a method for improving thedurability of sclero protein (e.g., brain meninges) by soaking thedegreased tissue in hydrogen peroxide or polyethylene glycol for severalhours prior to lyophilization. The resulting modified whole tissueexhibits increased persistence.

Hiroyoshi, U.S. Pat. No. 4,678,468, disclosed the preparation ofpolysiloxane polymers having an interpenetrating network ofwater-soluble polymer dispersed within. The water-soluble polymer may bea collagen derivative, and the polymer may additionally include heparin.The polymers are shaped into artificial blood vessel grafts, which aredesigned to prevent clotting.

Other patents disclose the use of collagen preparations incorporatingbone fragments or minerals. For example, Miyata et al, U.S. Pat. No.4,314,380, disclosed a bone implant prepared by baking animal bonesegments, then soaking the baked segments in a solution of atelopeptidecollagen. Deibig et al, U.S. Pat. No. 4,192,021, disclosed an implantmaterial which comprises powdered calcium phosphate in a pastyformulation with a biodegradable polymer (which may be collagen).Commonly owned U.S. application Ser. No. 06/855,004, filed Apr. 22,1986, now abandoned, disclosed a particularly effective bone repairmaterial comprising autologous bone marrow, collagen, and particulatecalcium phosphate in a solid, malleable formulation.

There are several references in the art to proteins modified by covalentconjugation to polymers to alter the solubility, antigenicity, andbiological clearance of the protein. For example, U.S. Pat. No.4,261,973 disclosed the conjugation of several allergens to PEG or PPG(polypropylene glycol) to reduce the proteins' immunogenicity. U.S. Pat.No. 4,301,144 disclosed the conjugation of hemoglobin with PEG and otherpolymers to increase the protein's oxygen-carrying capability. EPO98,110 disclosed coupling an enzyme or interferon to apolyoxyethylene-polyoxypropylene (POE-POP) block polymer to increase theprotein's half-life in serum. U.S. Pat. No. 4,179,337 disclosedconjugating hydrophilic enzymes and insulin to PEG or PPG to reduceimmunogenicity. Davis et al, Lancet (1981) 2:281-83, disclosed theenzyme uricase modified by conjugation with PEG to provide uric addmetabolism in serum having a long half-life and low immunogenicity.Nishida et al, J Pharm Pharmacol (1984) 36:354-55, disclosed PEG-uricaseconjugates administered orally to chickens, demonstrating decreasedserum levels of uric acid. Inada et al, Biochem & Biophys Res Comm(1984) 122:845-50 disclosed lipoprotein lipase conjugated with PEG torender it soluble in organic solvents. Takahashi et al, Biochem &Biophys Res Comm (1984) 121:261-65, disclosed HRP conjugated with PEG torender the enzyme soluble in benzene. Abuchowski et al, Cancer BiochemBiophys (1984) 7:175-86, disclosed that enzymes such as asparaginase,catalase, uricase, arginase, trypsin, superoxide dismutase, adenosinedeaminase, phenylalanine ammonia-lyase and the like conjugated with PEGexhibit longer half-lives in serum and decreased immunogenicity.However, these references are essentially concerned with modifying thesolubility and biological characteristics of proteins administered inlow concentrations in aqueous solution.

M. Chvapil et al, J Biomed Mater Res (1969) 3:315-32, disclosed acomposition prepared from collagen sponge and a crosslinked ethyleneglycol monomethacrylate-ethylene glycol dimethacrylate hydrogel. Thecollagen sponge was prepared by lyophilizing an aqueous mixture ofbovine hide collagen and methylglyoxal, a tanning agent. Thesponge-hydrogel composition was prepared by polymerizing ethylene glycolmonomethacrylate and ethylene glycol dimethacrylate in the sponge.

A series of related patents disclose various types ofcollagen-containing materials. The patents are U.S. Pat. No. 4,703,108,issued Oct. 27, 1987; U.S. Pat. No. 4,861,714, issued Aug. 29, 1989;U.S. Pat. No. 4,863,856, issued Sep. 5, 1989; U.S. Pat. No. 4,925,924,issued May 15, 1990; 4,970,298, issued Nov. 13, 1990; and U.S. Pat. No.4,997,753, issued Mar. 5, 1991. All of these patents disclose collagenmaterials wherein type I, II, and III collagens are contacted with acrosslinking agent selected from the group consisting of a carbodimideor a succinimidyl active ester. Various types of treatment may becarried out prior to or after crosslinking in order to form particulartypes of desired materials such as sponges and/or sheets.

In U.S. Pat. No. 5,162,430, we described chemical conjugates wherebyvarious forms of collagen were conjugated using synthetic hydrophilicpolymers such as polyethylene glycol. Such conjugates are useful for avariety of applications, such as soft tissue augmentation and theformation of implants useful in bone repair. In U.S. application Ser.No. 07/907,518, we disclose that it is possible to form such conjugateswith biomaterials other than collagen. Specifically, synthetichydrophilic polymers are used to crosslink insoluble biocompatible,biologically inert (preferably naturally occurring) polymers other thancollagen. Activated polyethylene glycol is the preferred crosslinkingagent. We now describe specific biocompatible polymer conjugates andtheir methods of synthesis, which include conjugates ofglycosaminoglycans, and/or their derivatives, which can be used in amanner similar to the collagen-polymer conjugates described in ourearlier, above-referenced U.S. Pat. No. 5,162,430, which is incorporatedherein by reference.

SUMMARY OF THE INVENTION

Biocompatible, pharmaceutically acceptable, nonimmunogenic conjugatesare formed by covalently binding glycosaminoglycans, and/or derivativesthereof, to a synthetic hydrophilic polymer, such as an activatedpolyethylene glycol, and optionally covalently binding the conjugate tocollagen.

The synthetic hydrophilic polymer is preferably an activatedpolyethylene glycol or a derivative thereof having an average molecularweight in the range of about 100 to about 100,000, preferably between1,500 to 20,000. Compositions comprising the conjugates may optionallyinclude other components such as pharmaceutically acceptable fluidcarriers to form injectable formulations, and/or biologically activeproteins such as cytokines or growth factors. The biocompatibleconjugates of the invention generally contain large amounts of waterwhen formed. The conjugates can be dehydrated to form relatively solidimplants for hard tissue augmentation, such as the repair or replacementof bone or cartilage. The dehydrated, solid implant can further be goundinto particles which can be suspended in a nonaqueous fluid and injectedinto a living being for soft tissue augmentation. Once in place, thesolid implants or particles rehydrate and expand in size five-fold ormore.

The invention relates to biocompatible conjugates which may be used in avariety of medical and pharmaceutical applications. The most basicembodiment includes the biocompatible conjugates and pharmaceuticalcompositions formulated using these conjugates, which may additionallyinclude pharmaceutically acceptable carriers in different types andamounts. The conjugates include a synthetic hydrophilic polymer, one ormore type of glycosaminoglycan, and optionally, collagen.

One of the most important uses for the conjugates and compositions ofthe invention is in soft tissue augmentation. The compositions areformulated into a flowable form and injected into patients, such as intofacial areas, for the purpose of soft tissue augmentation. The methodcan be varied so that the reaction between the glycosaminoglycan and thesynthetic polymer occurs in situ. Furthermore, the conjugates can bedehydrated and then ground into particles, suspended in an inertnonaqueous carrier, and injected into a patient. After injection, thecarrier will be removed by natural physiological conditions and theparticles will rehydrate and swell to their original size.

The conjugates can further be molded into a desired shape, thendehydrated to form a solid implant for use in hard tissue augmentation,such as for the repair or replacement of cartilage or bone.

The conjugates of the invention can be combined with cytokines or growthfactors. The cytokines can be either simply admixed with theglycosaminoglycan-synthetic polymer conjugate, or can be chemicallyconjugated to di- or multi-functionally activated polymer (e.g.,glycosaminoglycan-synthetic polymer-cytokine). In the case of anadmixture, the cytokines or growth factors are not chemically bound tothe conjugate and may diffuse out from the site of administration intothe surrounding tissue, providing for sustained release and localtherapeutic effects. In the case of the cytokine or growth factor beingchemically conjugated to the polymer conjugate, the cytokine or growthfactor retains its biological activity while bound to the conjugate andmay also be released by erosion of the polymer conjugate.

The conjugates of the invention, and compositions containing suchconjugates, are useful in a wide range of therapeutic applications. Forexample, the conjugates are useful in dermal wound healing andcardiovascular applications where immunological reactions are to beminimized or blood coagulation is to be avoided. The conjugates may alsobe used in various ophthalmic applications, such as vitreous fluidreplacement, corneal shields for delivery of drugs to the eye, or aslenticules. Other indications include use of the conjugates inorthopedic surgery or as joint lubricants in the treatment of arthritis.Other potential uses for the conjugates are as an injectable drug orcell delivery system, as a dermal would dressing, or as a coating forsolid implants intended for long-term use in the body.

The conjugates can further be made into a variety of forms, including,but not limited to, membranes, beads, sponges, tubes, sheets, and formedimplants. Formed implants can be used as prosthetic devices forreplacement or augmentation of various organs and body parts such asheart valves, patellas, ears, noses, cheekbones, etc.

A primary feature of the invention is to provide biocompatibleconjugates formed by covalently binding synthetic polymers such asactivated polyethylene glycol to one or more species ofglycosaminoglycan.

Another feature of the invention is to provideglycosaminoglycan-synthetic polymer conjugates which are furthercovalently bound to collagen.

Another feature of the invention is to provide pharmaceuticallyacceptable, nonimmunogenic compositions comprising pharmaceuticallyacceptable fluid carriers in which the conjugates are dispersed.

Another feature of the invention is to provide a method of tissueaugmentation comprising forming biocompatibleglycosaminoglycan-synthetic polymer conjugates, dehydrating theconjugates to form a solid, grinding the solid into particles,suspending the particles in a pharmaceutically acceptable nonaqueousfluid carrier, and injecting the suspension into the site ofaugmentation, after which the particles will rehydrate and expand insize.

An important advantage of the present invention is that theglycosaminoglycan-synthetic polymer conjugates have a greater degree ofstability in vivo as compared with conventional glycosaminoglycancompositions.

Another feature of the invention is that the glycosaminoglycan-syntheticpolymer conjugates can be formed using a range of different molecularweight synthetic polymers in order to adjust the physicalcharacteristics of the composition.

Another advantage of the present invention is that theglycosaminoglycan-synthetic polymer conjugates have superior handlingcharacteristics as compared with conventional glycosaminoglycancompositions.

Another advantage of the present invention is that theglycosaminoglycan-synthetic polymer conjugate compositions generate adecreased immune reaction as compared with conventional collagencompositions.

Another advantage of the present invention is that theglycosaminoglycan-synthetic polymer conjugate compositions have improvedmoldability, malleability, and elasticity as compared with conventionalglycosaminoglycan compositions.

Other features of the present invention include the ability to formulatethe compositions and conjugates in combination with pharmaceuticallyactive proteins such as cytokines or growth factors in order to improvethe activity and available half-life of such cytokines or growth factorsunder physiological conditions.

Another feature of the present invention is that the glycosaminoglycansor derivatives thereof may be bound to the synthetic polymer by means ofa variety of types of covalent linkages including ester and etherlinkages.

Another advantage of the present invention is that an ether linkage maybe used to form the covalent bond to create the conjugate and this bondis resistant to hydrolysis.

Another advantage of the invention is that the three-part conjugatescomprising covalently bonded glycosaminoglycan-syntheticpolymer-collagen have different physical and chemical properties thaneither glycosaminoglycan-synthetic polymer conjugates orcollagen-synthetic polymer conjugates alone, which properties can bemanipulated as desired by varying the relative ratios ofglycosaminoglycan and collagen in the composition.

These and other features of the present invention will become apparentto those skilled in the art upon reading the details of the structure,synthesis, and usage of the glycosaminoglycan-synthetic polymerconjugates set forth below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

It must be noted that, as used in this specification and the appendedclaims, the singular forms "a", "an", and "the" include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to "a polymer" includes mixtures of such polymers, referenceto "an attaching group or a linking group" includes one or moredifferent types of groups known by those skilled in the an or capable offorming a covalent bond, and reference to "the synthetic polymer"includes mixtures of different types of synthetic polymers such asvarious activated polyethlene glycols and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein may be usefulin the practice or testing of the present invention, only the preferredmethods and materials are described below; it is not inteneded that theinvention be limited to these preferred embodiments, however.

All publications mentioned herein are incorporated herein by reference.Further, specific terminology of particular importance to thedescription of the present invention is defined below.

Definitions

The term "glycosaminoglycan" is intended to encompass complexpolysaccharides which are not biologically active (i.e., not compoundssuch as ligands or proteins) having repeating units of either the samesaccharide subunit or two different saccharide subunits. Some examplesof glycosaminoglycans include dermatan sulfate, hyaluronic acid, thechondroitin sulfates, chitin, heparin, keratan sulfate, keratosulfate,and derivatives thereof. In general, the glycosaminoglycans areextracted from a natural source and purified and derivatized. However,they may be synthetically produced or synthesized by modifiedmicroorganisms such as bacteria.

The term "hyaluronic acid" is intended to encompass naturally occurringand synthetic forms of the polymer--(C₈ H₁₃ O₄ N)_(n).(C₆ H₈ O₅)_(n)O--(n=1 to n=about 5,000), and derivatives thereof. Particularlypreferred derivatives include those having functionalized moieties whichallow chemical reaction with another molecule to form a covalent bond,such as deacetylated hyaluronic acid. The compound includes alternatingunits of 1,4-linked N-acetylglucosamine and glucuronic acid units.Hyaluronic acid is a viscous, high molecular weight mucopolysaccharidefound in mammalian fluids and connective tissue. The formula forhyaluronic acid is shown below. ##STR1## wherein n ranges from 1 toabout 5,000.

The term "chondroitin sulfate", as used herein, is intended to encompassthree major compounds: chondroitin sulfate A, dermatan sulfate (alsoknown as chondroitin sulfate B, which is an isomer of chondroitinsulfate A), and chondroitin sulfate C. The structures of these threecompounds are shown below. ##STR2## wherein n ranges from about 10 toabout 300; ##STR3## wherein n ranges from about 20 to about 200;##STR4## wherein n ranges from about 10 to about 300.

The term "chitin" is intended to encompass polymers comprising repeatingunits of N-acetylglucosamine. The structure of chitin is shown below.##STR5## wherein n ranges from about 500 to about 2,000.

The term "chitosans" refers to both partially and fully deacetylatedchitins. The term "chitosan 1" refers to partially deacetylated chitin,as shown below. ##STR6## wherein n ranges from about 500 to about 2,000.

The term "chitosan 2" refers to fully deacetylated chitin, as shownbelow. ##STR7## wherein n ranges from about 500 to about 2,000.

The term "keratan sulfate" refers to polymers having the repeatingstructure shown below. ##STR8## wherein n ranges from about 10 to about100.

The term "keratosulfate" refers to a polymer that is an isomer ofkeratan sulfate, having the repeating structure shown below. ##STR9##wherein n ranges from about 10 to about 100.

The term "heparin" refers to polymers comprising alternating units ofsulfated glucosamine and sulfated glucuronic acid, as shown below.##STR10## wherein n ranges from about 2 to about 3,000.

The terms "biologically inert polymers", "biocompatible polymers", and"biologically inert, biocompatible polymers" are used interchangeablyherein. The terms refer to biologically inert, insoluble, biocompatiblepolymers and their derivatives which can be covalently bound tosynthetic hydrophilic polymers to form the conjugates of the invention.These terms encompass polymers that are biologically inert, insoluble,nontoxic and do not generate any appreciable immune reaction whenincorporated into a living being such as a human.

Preferred synthetic polymers for use in the present invention arehydrophilic and are highly pure or are purified to a highly pure statesuch that the polymer is or is treated to become pharmaceutically pureso that it may be injected into a human patient. Most hydrophilicsynthetic polymers can be rendered water-soluble by incorporating asufficient number of oxygen (or less frequently nitrogen) atomsavailable for forming hydrogen bonds in aqueous solutions. Preferredsynthetic polymers are hydrophilic but not necessarily water-soluble.Hydrophilic synthetic polymers used herein include activated forms ofpolyethylene glycol (PEG), polyoxyethylene, polymethylene glycol,polytrimethylene glycols, polyvinylpyrrolidones, and derivatives thereofwith activated PEG being particularly preferred. The synthetic polymerscan be linear or multiply branched, but are typically not substantiallycrosslinked. Other suitable hydrophilic synthetic polymers includepolyoxyethylene-polyoxypropylene block polymers and copolymers.Polyoxyethylene-polyoxypropylene block polymers having an ethylene dimenucleus (and thus having four ends) are commercially available and maybe used in the practice of the invention. Naturally occurring polymerssuch as proteins, starch, cellulose, heparin, hyaluronic acid andderivatives thereof and the like are expressly excluded from the scopeof this definition. All suitable synthetic polymers will be non-toxic,non-inflammatory, and nonimmunogenic when administered subcutaneously,and will preferably be essentially nondegradable in vivo over a periodof at least several months. The hydrophilic synthetic polymer mayincrease the hydrophilicity of the conjugate, but does not render itwater-soluble. The most preferred hydrophilic synthetic polymers includemono-, di-, and multifunctionally activated polyethylene glycols.Monofunctionally activated PEG has only one reactive hydroxy group,while difunctionally activated PEG has reactive groups at each end.Monofunctionally activated PEG preferably has an average molecularweight between about 100 and about 15,000, more preferably between about200 and about 8,000, and most preferably about 5,000. Difunctionallyactivated PEG preferably has an average molecular weight of about 400 toabout 40,000, more preferably between about 3,000 to about 10,000.Multifunctionally activated PEG preferably has an average molecularweight between about 3,000 and 100,000.

PEG can be rendered monofunctionally activated by forming an alkyleneether group at one end. The alkylene ether group may be any suitablealkoxy radical having 1-6 carbon atoms, for example, methoxy, ethoxy,propoxy, 2-propoxy, butoxy, hexyloxy, and the like. Methoxy is presentlypreferred. Difunctionally activated PEG is provided by allowing areactive hydroxy group at each end of the linear molecule. The reactivegroups are preferably at the ends of the polymer, but may be providedalong the length thereof. Multifunctionally activated synthetic polymersare capable of crosslinking the compositions of the invention, and maybe used to attach cytokines or growth factors to theglycosaminoglycan-synthetic polymer conjugate.

The term "nonimmunogenic" refers to molecules and compositions whichproduce no appreciable immunogenic or allergic reaction when injected orotherwise implanted into the body of a human subject.

The term "chemically conjugated" as used herein means attached through acovalent chemical bond. In the practice of the invention, a hydrophilicsynthetic polymer and a glycosaminoglycan or derivative thereof may bechemically conjugated by using a linking radical, so that thehydrophilic synthetic polymer and glycosaminoglycan are each bound tothe radical, but not directly to each other. The term "biocompatibleconjugate" refers to a biologically inert, biocompatible polymerchemically conjugated to a hydrophilic synthetic polymer, within themeaning of this invention. For example,"PEG-hyaluronic acid" denotes acomposition of the invention wherein hyaluronic acid is chemicallyconjugated to PEG. The hydrophilic synthetic polymer may be "chemicallyconjugated" to the glycosaminoglycan such as hyaluronic acid by means ofa number of different types of chemical linkages. For example, theconjugation can be via an ester or a urethane linkage, but is morepreferably by means of an ether linkage. An ether linkage is preferredin that it can be formed without the use of toxic chemicals and is notreadily susceptible to hydrolysis in vivo.

Those of ordinary skill in the art will appreciate that hydrophilicsynthetic polymers such as polyethylene glycol cannot be preparedpractically to have exact molecular weights, and that the term"molecular weight" as used herein refers to the average molecular weightof a number of molecules in any given sample, as commonly used in theart. Thus, a sample of PEG 2,000 might contain a statistical mixture ofpolymer molecules ranging in weight from, for example, 1,500 to 2,500,with one molecule differing slightly from the next over a range.Specification of a range of molecular weight indicates that the avengemolecular weight may be any value between the limits specified, and mayinclude molecules outside those limits. Thus, a molecular weight rangeof about 800 to about 20,000 indicates an average molecular weight of atleast about 800, ranging up to about 20,000.

The term "available lysine residue" as used herein refers to lysine sidechains exposed on the outer surface of natural polymer molecules, whichare positioned in a manner to allow reaction with activated PEG. Thenumber of available lysine residues may be determined by reaction withsodium 2,4,6-trinitrobenzenesulfonate (TNBS).

The terms "treat" and "treatment" as used herein refer to augmentation,repair, prevention, or alleviation of defects, particularly defects dueto loss or absence of soft tissue or soft tissue support, or to loss orabsence of bone. Additionally, "treat" and "treatment" also refer to theprevention, maintenance, or alleviation of disorders or disease using abiologically active protein coupled to a conjugate-containingcomposition of the invention. Accordingly, treatment of soft tissueincludes augmentation of soft tissue, for example, implantation ofconjugates of the invention to restore normal or desirable dermalcontours, as in the removal of dermal creases or furrows, or as in thereplacement of subcutaneous fit in maxillary areas where the fat is lostdue to aging, or in the augmentation of submucosal tissue, such as theurinary or lower esophageal sphincters. Treatment of bone and cartilageincludes the use of biocompatible conjugates, particularly incombination with suitable particulate materials, to replace or repairbone tissue, for example, in the treatment of bone nonunions orfractures. Treatment of bone also includes use of conjugate-containingcompositions, with or without additional bone growth factors.Compositions comprising conjugates with ceramic particles, preferablyhydroxyapatite and/or tricalcium phosphate, are particularly useful forthe repair of stress-bearing bone due to its high tensile strength.

The terms "cytokine" and "growth factor" are used to describebiologically active molecules and active peptides (which may be eithernaturally occurring or synthetic) which aid in healing or regrowth ofnormal tissue including growth factors and active peptides. The functionof cytokines is two-fold: 1) they can incite local cells to produce newcollagen or tissue, or 2) they can attract cells to the site in need ofcorrection. As such, cytokines and growth factors serve to encourage"biological anchoring" of the implant within the host tissue. Aspreviously described, the cytokines can either be admixed with theconjugate or chemically coupled to the conjugate. For example, one mayincorporate cytokines such as interferons (IFN), tumor necrosis factors(TNF), interleukins, colony stimulating factors (CSFs), or growthfactors such as osteogenic factor extract (OFE), epidermal growth factor(EGF), transforming growth factor (TGF) alpha, TGF-β (including anycombination of TGF-βs), TGF-β1, TGF-β2, platelet derived growth factor(PDGF-AA, PDGF-AB, PDGF-BB), acidic fibroblast growth factor (FGF),basic FGF, connective tissue activating peptides (CTAP),β-thromboglobulin, insulin-like growth factors, erythropoietin (EPO),nerve growth factor (NGF), bone morphogenic protein (BMP), osteogenicfactors, and the like. Incorporation of such cytokines, and appropriatecombinations of cytokines and growth factors can facilitate the regrowthand remodeling of the implant into normal tissue, or may be used in thetreatment of wounds. Further, one may chemically link the cytokines orgrowth factors to the glycosaminoglycan-synthetic polymer conjugate byemploying a suitable amount of multifunctionally activated syntheticpolymer molecules during synthesis. The cytokines or growth factors maythen be attached to the functional sites of the multifunctionallyactivated synthetic polymers by the same method used to attach activatedPEG to glycosaminoglycans, or by any other suitable method. By tetheringcytokine or growth factor molecules to the implant, the amount ofcytokines or growth factor required to be therapeutically effective issubstantially reduced. Conjugates incorporated with cytokines or growthfactors may serve as effective controlled release drug delivery means.By varying the chemical linkage between the glycosaminoglycan and thesynthetic polymer, it is possible to vary the effect with respect to therelease of the cytokine or growth factor. For example, when an "ester"linkage is used, the linkage is more easily broken under physiologicalconditions, allowing for sustained release of the growth factor orcytokine from the matrix. However, when an "ether" linkage is used, thebonds are not easily broken and the cytokine or growth factor willremain in place for longer periods of time with its active sitesexposed, providing a biological effect on the natural substrate for theactive site of the protein. It is possible to include a mixture ofconjugates with different linkages so as to obtain variations in theeffect with respect to the release of the cytokine or growth factor,i.e., the sustained release effect can be modified to obtain the desiredrate of release.

The term "effective amount" refers to the amount of composition requiredin order to obtain the effect desired. Thus, a "tissue growth-promotingamount" of a composition containing a cytokine or growth factor refersto the amount of cytokine or growth factor needed in order to stimulatetissue growth to a detectable degree. Tissue, in this context, includesconnective tissue, bone, cartilage, epidermis and dermis, blood, andother tissues. The actual mount which is determined to be an effectivemount will vary depending on factors such as the size, condition, sexand age of the patient and can be more readily determined by thecaregiver.

The term "sufficient mount" as used herein is applied to the amount ofcarder used in combination with the conjugates of the invention. Asufficient mount is that mount which, when mixed with the conjugate,renders it in the physical form desired, for example, injectablesolution, injectable suspension, plastic or malleable implant, rigidstress-bearing implant, and so forth. Injectable formulations generallyinclude an amount of fluid carrier sufficient to render the compositionsmoothly injectable, whereas malleable implants have substantially lesscarrier and have a clay-like consistency. Rigid stress-bearing implantsmay include no carrier at all and have a high degree of structuralintegrity. The amount of the carrier can be varied and adjusteddepending on the particular conjugate used and the end result desired.Such adjustments will be apparent to those skilled in the art uponreading this disclosure.

The term "suitable particulate material" as used herein refers to aparticulate material which is substantially insoluble in water,nonimmunogenic, biocompatible, and immiscible with collagen-polymerconjugate. The particles of material may be fibrillar, or may range insize from about 20 to 250 μm in diameter and be bead-like or irregularin shape. Exemplary particulate materials include without limitationfibrillar crosslinked collagen, gelatin beads, crosslinked collagen-dPEGparticles, polytetrafluoroethylene beads, silicone rubber beads,hydrogel beads, silicon carbide beads, and glass beads. Preferredparticulate materials are calcium phosphates, most preferablyhydroxyapatite and/or tricalcium phosphate particles.

The term "solid implant" refers to any solid object which is designedfor insertion and use within the body, and includes bone and cartilageimplants (e.g., artificial joints, retaining pins, cranial plates, andthe like, of metal, plastic and/or other materials), breast implants(e.g., silicone gel envelopes, foam forms, and the like), catheters andcannulas intended for long-term use (beyond about three days),artificial organs and vessels (e.g., artificial hearts, pancreases,kidneys, blood vessels, and the like), drug delivery devices (includingmonolithic implants, pumps and controlled release devices such as Alzet®minipumps, steroid pellets for anabolic growth or contraception, and thelike), sutures for dermal or internal use, periodontal membranes,lenticules, corneal shields, platinum wires for aneurysm treatment, andthe like.

The term "suitable fibrous material", as used herein, refers to afibrous material which is substantially insoluble in water,nonimmunogenic, biocompatible, and immiscible with the biocompatibleconjugate of the invention. The fibrous material may comprise a varietyof materials having these characteristics and are combined withcompositions of the conjugate in order to form and/or provide structuralintegrity to various implants or devices used in connection with medicaland pharmaceutical uses. For example, the conjugate compositions of theinvention can be coated on the "suitable fibrous material" which canthen be wrapped around a bone to provide structural integrity to thebone. Thus, the "suitable fibrous material" is useful in forming the"solid implants" of the invention.

The term "in situ" as used herein means at the site of administration.Thus, the injectable reaction mixture compositions are injected orotherwise applied to a site in need of augmentation, and allowed tocrosslink at the site of injection. Suitable sites will generally beintradermal or subcutaneous regions for augmenting dermal support, atthe site of bone fractures for wound healing and bone repair, and withinsphincter tissue for sphincter augmentation (e.g., for restoration ofcontinence).

The term "aqueous mixture" includes liquid solutions, suspensions,dispersions, colloids, and the like containing a natural polymer andwater.

The term "collagen" is used in its conventional sense to describe amaterial which is the major protein component of the extracellularmatrix of bone, cartilage, skin, and connective tissue in animals andderivatives. Collagen in its native form is typically a rigid,rod-shaped molecule approximately 300 nm long and 1.5 nm in diameter. Itis composed of three collagen polypeptides which form a tight triplehelix. The collagen polypeptides are characterized by a long midsectionhaving the repeating sequence -Gly-X-Y-, where X and Y are often prolineor hydroxyproline, bounded at each end by the "telopeptide" regions,which constitute less than about 5% of the molecule. The telopeptideregions of the collagen chains are typically responsible for thecrosslinking between chains, and for the immunogenicity of the protein.Collagen occurs in several "types", having differing physicalproperties. The most abundant types are Types I-III. The presentdisclosure includes these and other known types of collagen includingnatural collagen and collagen which is processed or modified, i.e.,various collagen derivatives. Collagen is typically isolated fromnatural sources, such as bovine hide, cartilage, or bones. Bones areusually dried, defatted, crushed, and demineralized to extract collagen,while hide and cartilage are usually minced and digested withproteolytic enzymes (other than collagenase). As collagen is resistantto most proteolytic enzymes, this procedure conveniently serves toremove most of the contaminating protein found with collagen.

The term "dehydrated" means the material is air-dried or lyophilized toremove substantially all unbound water.

General Method

To form the conjugates of the invention, glycosaminoglycans are, ingeneral, chemically derivatized and then covalently bound to afunctionally activated hydrophilic synthetic polymer. This can becarried out using a number of suitable methods. In accordance with onemethod, (1) the hydrophilic synthetic polymer is activated, (2) theglycosaminoglycan is subjected to chemical modification by deacetylationand/or desulfation, and (3) the activated synthetic polymer is reactedwith the chemically modified glycosaminoglycan.

Activation of Polyethylene Glycol (PEG)

The first step in forming the conjugates of the invention generallyinvolves functionalization, or activation, of the synthetic hydrophilicpolymer. Although different synthetic hydrophilic synthetic polymers canbe used in connection with forming the conjugate, the polymer must bebiocompatible, hydrophilic, but relatively insoluble, and is preferablyone or more forms of derivatized polyethylene glycol (PEG), due to itsknown biocompatibility. Various forms of derivatized PEG are extensivelyused in the modification of biologically active molecules because PEGcan be formulated to have a wide range of solubilities and because itlacks toxicity, antigenicity, immunogenicity, and does not typicallyinterfere with the enzymatic activities and/or conformations ofpeptides. Furthermore, PEG is generally non-biodegradable and is easilyexcreted from most living organisms including humans.

Various functionalized polyethylene glycols have been used effectivelyin fields such as protein modification (see Abuchowski et al., Enzymesas Drugs, John Wiley & Sons: New York, N.Y. (1981) pp. 367-383; andDreborg et al., Crit. Rev. Therap. Drug Carrier Syst. (1990) 6:315),peptide chemistry (see Mutter et al., The Peptides, Academic: New York,N.Y. 2:285-332; and Zalipsky et al., Int. J. Peptide Protein Res. (1987)30:740), and the synthesis of polymeric drugs (see Zalipsky et al., Eur.Polym. J. (1983) 19:1177; and Ouchi et al., J. Macromol. Sci.-Chem.(1987) A24:1011). Various types of conjugates formed by the binding ofactivated (functionalized) polyethylene glycol with specificpharmaceutically active proteins have been disclosed and found to haveuseful medical applications in part due to the stability of suchconjugates with respect to proteolytic digestion, reducedimmunogenicity, and longer half-lives within living organisms.

One form of polyethylene glycol which has been found to be particularlyuseful is monomethoxy-polyethylene glycol (mPEG), which can be activatedby the addition of a compound such as cyanuric chloride, then coupled toa protein (see Abuchowski et al., J. Biol. Chem. (1977) 252:3578).Although such methods of activating polyethylene glycol can be used inconnection with the present invention, they are not particularlydesirable in that the cyanuric chloride is relatively toxic and must becompletely removed from any resulting product in order to provide apharmaceutically acceptable composition.

Activated forms of PEG, including activated forms of mPEG, can be madefrom reactants which can be purchased commercially. One form ofactivated PEG which has been found to be particularly useful inconnection with the present invention ismPEG-succinate-N-hydroxysuccinimide ester (SS-PEG) (see Abuchowski etal., Cancer Biochem. Biphys. (1984) 7:175). Activated forms of PEG suchas SS-PEG react with the proteins under relatively mild conditions andproduce conjugates without destroying the specific biological activityand specificity of the protein attached to the PEG. However, when suchactivated PEGs are reacted with proteins, they react and form linkagesby means of ester bonds. Although ester linkages can be used inconnection with the present invention, they are not particularlypreferred in that they undergo hydrolysis when subjected tophysiological conditions over extended periods of time (see Dreborg etal., Crit. Rev. Therap. Drug Carrier Syst. (1990) 6:315; and Ulbrich etal., J. Makromol. Chem. (1986) 187:1131).

It is possible to link PEG to proteins via urethane linkages, therebyproviding a more stable attachment which is more resistant to hydrolyticdigestion than the ester linkages (see Zalipsky et al., Polymeric Drugand Drug Delivery Systems, Chapter 10, "Succinimidyl Carbonates ofPolyethylene Glycol" (1991)). The stability of urethane linkages hasbeen demonstrated under physiological conditions (see Veronese et al.,Appl. Biochem. Biotechnol. (1985) 11:141; and Larwood et al., J.Labelled Compounds Radiopharm. (1984) 21:603). Another means ofattaching the PEG to a protein can be by means of a carbamate linkage(see Beauchamp et al., Anal. Biochem. (1983) 131:25; and Berger et al.,Blood (1988) 71:1641). The carbamate linkage is created by the use ofcarbonyldiimidazole-activated PEG. Although such linkages haveadvantages, the reactions are relatively slow and may take 2 to 3 daysto complete.

The various means of activating PEG described above and publicationscited in connection with the activation means are described inconnection with linking the PEG to specific biologically active proteinsand not inert, biologically inactive, natural polymers. (See PolymericDrug and Drug Delivery Systems, Chapter 10, "Succinimidyl Carbonates ofPolyethylene Glycol" (1991 ).) However, the present invention nowdiscloses that such activated PEG compounds can be used in connectionwith the formation of inert, biocompatible conjugates. Such conjugatesprovide a range of improved, unexpected characteristics and as such canbe used to form the various compositions of the present invention.

Specific Forms of Activated PEG

For use in the present invention, polyethylene glycol is modified inorder to provide activated groups on one or, preferably, two or moreends of the molecule so that covalent binding can occur between the PEGand the free amino groups on the chemically derivatizedglycosaminoglycan. Some specific activated forms of PEG are shownstructurally below, as are generalized reaction products obtained byreacting activated forms of PEG with derivatized glycosaminoglycans. InFormulas 1-7, the term GAG-PLYM is used to represent chemicallyderivatized glycosaminoglycan polymers.

The first activated PEG is difunctionally activated PEG succinimidylglutarate, referred to herein as (SG-PEG). The structural formula ofthis molecule and the reaction product obtained by reacting it with aglycosaminoglycan derivative are shown in Formula 1. ##STR11##

Another difunctionally activated form of PEG is referred to as PEGsuccinimidyl (S-PEG). The structural formula for this compound and thereaction product obtained by reacting it with a glycosaminoglycanderivative such as deacetylated hyaluronic acid is shown in Formula 2.In any general structural formula for the compounds, the subscript 3 isreplaced with an "n" . In the embodiment shown in Formula 1, n=3, inthat there are three repeating CH₂ groups on either side of the PEG. Thestructure in Formula 2 results in a conjugate which includes an "ether"linkage which is not subject to hydrolysis. This is distinct from theconjugate shown in Formula 1, wherein an ester linkage is provided. Theester linkage is subject to hydrolysis under physiological conditions.##STR12##

Yet another difunctionally activated form of polyethylene glycol,wherein n=2, is shown in Formula 3, as is the conjugate formed byreacting the activated PEG with a glycosaminoglycan derivative.##STR13##

Another preferred embodiment of the invention similar to the compoundsof Formulas 2 and 3 is provided when n=l. The structural formula andresulting conjugate are shown in Formula 4. It is noted that theconjugate includes both an ether and a peptide linkage. These linkagesare stable under physiological conditions. ##STR14##

Yet another difunctionally activated form of PEG is provided when n=0.This compound is referred to as PEG succmimidyl carbonate (SC-PEG). Thestructural formula of this compound and the conjugate formed by reactingSC-PEG with a glycosaminoglycan derivative is shown in Formula 5.##STR15##

All of the derivatives depicted in Formulas 1-5 involve the inclusion ofthe succinimidyl group. However, different activating groups can beattached to one or both ends of the PEG molecule. For example, PEG canbe derivatized to form difunctionally activated PEG propion aldehyde(A-PEG), which is shown in Formula 6, as is the conjugate formed by thereaction of A-PEG with a glycosaminoglycan derivative. The linkage shownin Formula 6 is referred to as a --(CH₂)_(n) --NH-- linkage, wheren=1-10. ##STR16##

Yet another difunctionally activated form of polyethylene glycol is PEGglycidyl ether (E-PEG), which is shown in Formula 7, as is the conjugateformed by reacting such with a glycosaminoglycan derivative. ##STR17##

Chemical Derivatization of Glycosaminoglycans

To make the glycosaminoglycan-polymer conjugates of the presentinvention, the glycosaminoglycan first must be chemically derivatized ina manner that will provide free amino (--NH₂) groups which are availablefor covalent crosslinking with PEG. Chemical derivatization of theglycosaminoglycan to provide free amino groups can be accomplished byeither deacetylation of desulfation, both of which may be effected bythe addition of a strong base such as sodium hydroxide to theglycosaminoglycan solution.

Glycosaminoglycans such as hyaluronic acid, the chondroitin sulfates,keratan sulfate, keratosulfate, and chitin can be deacetylated (removalof the --COCH₃ group) to provide free amino groups, as shown in ReactionSchemes 1 and 2 for hyaluronic acid and chitin, respectively. ##STR18##

Glycosaminoglycans such as heparin, the chondroitin sulfates, keratansulfate, and keratosulfate can be desulfated (removal of the --SO₃group) to provide free amino groups, as shown in Reaction Scheme 3.##STR19##

As per Table 1, below, certain glycosaminoglycans, such as thechondroitin sulfates, keratan sulfate, and keratosulfate contain both--COCH₃ and --SO₃ groups and are therefore subject to both deacetylationand desulfation by the addition of sodium hydroxide. Deacetylation anddesulfation of chondroitin sulfate C is shown in Reaction Scheme 4.

                  TABLE 1                                                         ______________________________________                                        Derivatization of Glycosaminoglycans by Deacetylation                         and Desulfation                                                               Compound         Deacetylation                                                                            Desulfation                                       ______________________________________                                        Chitin           Yes        No                                                Chondroitin sulfate A                                                                          Yes        Yes                                               Chondroitin sulfate B                                                                          Yes        Yes                                               Chondroitin sulfate C                                                                          Yes        Yes                                               Heparin          No         Yes                                               Hyaluronic acid  Yes        No                                                Keratan sulfate  Yes        Yes                                               Keratosulfate    Yes        Yes                                               ______________________________________                                    

Crosslinking of Chemically Derivatized Glycosaminoglycans with PEG

Glycosaminoglycans that have been chemically derivatized to have freeamino groups can be crosslinked with activated multifunctional PEG, asshown in Reaction Scheme 5 for deacetylated hyaluronic acid. ##STR20##

Glycosaminoglycan-polymer conjugates are formed within minutes ofcombining the chemically derivatized glycosaminoglycan and thefunctionally activated polymer. The glycosaminoglycan derivative can bemixed with the polymer using syringe-to-syringe mixing. Alternatively,the glycosaminoglycan derivative can be extruded into a solution of theactivated polymer, crosslinking will occur as the polymer diffuses intothe glycosaminoglycan.

The rate of conjugate formation and the characteristics of the resultingconjugate can be varied by varying the type of activated PEG used and/orthe molecular weight and concentration of the PEG. In general, the useof PEG species (such as S-PEG) which result in ether or urethanelinkages lead to the creation of more stable conjugates than those whichresult in the readily hydrolyzed ester linkages. However, in certainsituations, such as drug delivery applications, it is desirable toinclude the weaker ester linkages: the linkages are gradually broken byhydrolysis under physiological conditions, breaking apart the matrix andreleasing the pharmaceutically active component held therein. Differentspecies of PEG can be mixed and used in the same drug deliverycomposition, resulting in a varied rate of matrix degradation and,hence, drug release.

Multifunctionally activated PEG can be used to crosslink more than onespecies of glycosaminoglycan derivative, or glycosaminoglycanderivatives and collagen, as shown in Reaction Scheme 6 for deacetylatedhyaluronic acid and collagen. The resulting composite material hasdifferent physical and chemical properties than either PEG-crosslinkedcollagen or PEG-crosslinked glycosaminoglycan alone. ##STR21##

The glycosaminoglycan-polymer-collagen composites can be produced in anumber of ways, as described in experimental Examples 3-6.

Suitable collagens for use in the invention include all types ofcollagen; however, types I, II, and III are preferred. The collagen usedin the practice of the invention may be either fibrillar (e.g., Zyderm®Collagen) or nonfibrillar. Either atelopeptide or telopeptide-containingcollagen may be used, depending on the desired end use of the conjugate.Various forms of collagen are available commercially, or may be preparedby the processes described in, for example, U.S. Pat. Nos. 3,949,073;4,488,911; 4,424,208; 4,582,640; 4,642,117; 4,557,764; and 4,689,399,all incorporated herein by reference.

Collagen contains a number of available amino and hydroxy groups whichmay be used to bind the collagen to the glycosaminoglycan-syntheticpolymer conjugate. Methods of conjugating collagen to polyethyleneglycol are discussed in detail in U.S. Pat. No. 5,162,430.

Use and Administration

The primary use of the glycosaminoglycan-synthetic polymer andglycosaminoglycan-synthetic polymer-collagen conjugates of the inventionis as injectable compositions for soft tissue augmentation (such asdermal augmentation or sphincter augmentation) and drug delivery. Forinjectable formulations, glycosaminoglycan concentrations within therange of about 10 to about 100 mg/mL are generally used. Theconcentration of activated synthetic polymer in the composition ispreferably within the range of about 1 to about 400 milligrams ofactivated synthetic polymer per millileter of composition.

Crosslinking between the glycosaminoglycan and the synthetic polymer canbe performed in vitro, or a reaction mixture may be injected forcrosslinking in situ. The glycosaminoglycan derivative and activatedpolymer can be stored in separate barrels of a double-barreled syringe.As the plunger of the syringe is depressed and the material is injectedbeneath the skin, the components mix in the needle of syringe andcrosslink in situ. Some of the activated polymer molecules mayadditionally crosslink to the patient's own collagen to anchor theimplant in place. Gel formation will occur within twenty minutes or lessof administration. Injectable compositions may further be used for hardtissue repair in situations where surgery is not desirable orrecommended. In hard tissue applications, the injectable compositionserves as a matrix for regeneration of bone or cartilage at the site ofplacement.

In addition to aqueous injectable solutions, prepolymerizedglycosaminoglycan-polymer conjugates can be dried and then ground intodried particulates. Alternatively, glycosaminoglycan-polymer conjugatescan be dried in bead or droplet form. The beads or particles comprisingthe conjugates can be suspended in a nonaqueous carrier and injected toa soft tissue site in need of augmentation. Once in situ, theparticulates rehydrate and swell three- to five-fold due to thehydrophilicity of the polyethylene glycol molecules. Less volume ofproduct is therefore required to achieve the desired connection.

The multifunctionally activated synthetic polymers may be used tocovalently crosslink glycosaminoglycan derivatives to collagen or tobiologically active proteins such as cytokines and growth factors. Suchcompositions are particularly suited for use in wound healing,osteogenesis, and immune modulation. Tethering of biologically activemolecules to glycosaminoglycans provides an effective sustained releasedrug delivery system. As described above, different species ofpolyethylene glycol can be included in the formulation to result invarying rates of drug release.

Compositions of the invention containing biologically active cytokinesor growth factors such as TGF-β are prepared by admixing an appropriateamount of the cytokine or growth factor into the composition, or byincorporating the cytokine or growth factor into the glycosaminoglycanprior to treatment with activated PEG. Preferably, the cytokine orgrowth factor is first reacted with a molar excess of amultifunctionally activated polyethylene glycol in a dilute solution forthree to four minutes. The cytokine or growth factor is preferablyprovided at a concentration of about 1 μg/mL to about 5 mg/mL, while theactivated polymer is preferably added to a final concentration providinga thirty- to fifty-fold molar excess. The conjugated biologically activefactor-synthetic polymer is then added to an aqueous glycosaminoglycanmixture (preferably having a concentration within the range of about 1to about 60 mg/mL) at neutral pH (approximately 7-8) and allowed toreact further to form biologically active factor-syntheticpolymer-glycosaminoglycan conjugates. The resulting composition isallowed to stand overnight at ambient temperature. The pellet iscollected by centrifugation and washed with PBS, using vigorousvortexing to remove unbound factor.

Compositions of the invention containing biologically active factorssuch as cytokines or growth factors are particularly suited forsustained release of factors, as in the case of wound healing promotion.Osteoinductive factors and cofactors (including TGF-β) and bonemorphogenic protein (BMP) may advantageously be incorporated intocompositions for bone replacement, augmentation, and/or defect repair.Alternatively, one may administer antiviral and antitumor factors suchas TNF, interferons, CSFs, TGF-β, and the like for their pharmaceuticalactivities. The amount of cytokine or growth factor incorporated intothe composition is determined based on the type of factor being used,the severity of the condition being treated, the rate of deliverydesired, etc. These parameters may be determined by routineexperimentation; for example, by preparing a conjugatedfactor-polymer-glycosaminoglycan composition as described above andassaying the release rate of factor in a suitable animal model.

Compositions of glycosaminoglycan-synthetic polymer conjugates can alsobe formed into relatively solid implants. Compositions of the inventioncan be prepared in a form that is dense and rigid enough to substitutefor cartilage. These compositions are useful for repairing andsupporting tissues which require some degree of structure and rigidity,for example, in reconstruction of the nose, ear, knee, larynx, trachealrings, and joint surfaces. One can also replace tendons, ligaments, andblood vessels using appropriately formed cartilaginoid materials. Inthese applications, the material is generally cast or molded into thedesired shape. Materials for tendon and ligament replacement may beformed by braiding or weaving filaments of theglycosaminoglycan--polymer conjugates into cords or ropes. In the caseof artificial blood vessels, it may be advantageous to incorporate areinforcing mesh (e.g., nylon, Teflon®, or Dacron®).

Formulations suitable for repair of bone defects or nonunions may beprepared by providing high concentration compositions of biocompatibleconjugates, such as glycosaminoglycan-synthetic polymer;glycosaminoglycon-synthetic polymer-collagen; or one of these conjugatesin combination with a cytokine or growth factor, any of which may beused in admixture with suitable particulate materials. When making bonerepair compositions intended to persist for long periods of time invivo,the linkage between the glycosaminoglycan and synthetic polymer may bean ether linkage in order to avoid deterioration due to the hydrolysisof the ester linkages. Such conjugate/particulate compositions may bemalleable or rigid, depending on the amount of liquid incorporated.Formulations for treatment of stress-bearing bone are preferably driedand rigid, and will generally comprise between about 45% and 85%particulate mineral, for example, hydroxyapatite or tricalciumphosphate, or mixtures thereof. The tensile strength and rigidity may befurther increased by heating the composition under vacuum at about60°-90° C., preferably about 75° C., for about 5 to 15 hours, preferablyabout 10 hours.

Flexible sheets or membranous forms of glycosaminoglycan-syntheticpolymer conjugates may be prepared by methods known in the art; forexample, U.S. Pat. Nos. 4,600,533, 4,412,947, and 4,242,291. Briefly, anaqueous solution of glycosaminoglycan having a concentration in therange of approximately 10-100 mg/mL is east into the bottom of a flatcontainer. A solution of activated polyethylene glycol is added to theglycosaminoglycan solution and allowed to react at room temperature fora period of time ranging from several hours to overnight. The resultingglycosaminoglycan-polymer conjugate is removed from the bottom of thecontainer using a spatula and then washed with PBS to remove excessunreacted polymer.

The resulting conjugate composition may be compressed under constantpressure to form a uniform sheet or mat and optionally dehydrated undera vacuum oven or by lyophilization or air-drying to form a membrane ofthe invention. More flexible membranes can be obtained using lowerglycosaminoglycan concentrations, higher synthetic polymerconcentrations, and shorter reaction times.

Glycosaminoglycan-synthetic polymer conjugates may also be prepared inthe form of sponges by lyophilizing an aqueous slurry of the compositionafter conjugation.

Glycosaminoglycan-synthetic polymer conjugate compositions can beformulated into hydrogels having moisture contents in the range of about5 to about 95%. By varying the moisture content, hydrogels of varyingdensity and stiffness may be obtained, depending on the desired end useapplication.

Glycosaminoglycan-synthetic polymer conjugates can be used to coatbreast implants. The surface of a standard silicone shell implant can bechemically derivatized to provide active binding sites for di- ormultifunctionally activated PEG-glycosaminoglycan(glycosaminoglycan-PEG-silicone). The presence of the conjugate coatingbound directly to the silicone via activated PEG will reduce scar tissueformation and capsular contracture. Unlike typical coated implants, scartissue will not be able to grow between the coating and the implantbecause the coating is conjugated directly to the surface of theimplant.

Alternatively, a flexible sheet of glycosaminoglycan-synthetic polymerconjugate formulation can be formed into a hollow sphere for use as abreast implant shell. The shell can then be filled with a radiolucentmaterial, such as a triglyceride, to facilitate mammography.

Formulations of glycosaminoglycan-synthetic polymer conjugates may alsobe used to coat other types of implants for long-term use in the body,such as catheters, cannulas, bone prostheses, cartilage replacements,minipumps and other drug delivery devices, artificial organs and bloodvessels, meshes for tissue reinforcement, etc.Glycosaminoglycan-synthetic polymer compositions can also be used tocoat platinum wires, which can then be administered to the site of ananeurysm via catheter. Such surface treatment renders the implantsnonimmunogenic and reduces the incidence of foreign body reaction.

Coating of an implant with a conjugate composition may be accomplishedby dipping the implant into a solution containing glycosaminoglycan andsynthetic polymer while crosslinking is occurring and allowing theadherent viscous coating to dry as crosslinking is completed. One maypour, brush, or otherwise apply the reaction mixture to the implant ifdipping is not feasible. Alternatively, one may use flexible sheets ormembranous forms of the conjugate to wrap the object, sealing comers andedges with reaction mixture.

EXAMPLES

The following examples are set forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the conjugates and formulations and implants containing suchconjugates and are not intended to limit the scope of the invention.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, molecular weight, etc.), but someexperimental errors and deviation should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isaverage molecular weight, temperature is in degrees Centigrade, andpressure is at or near atmospheric.

Example 1

One (1) gram of sodium hyaluronate (obtained from LifeCore Biomedical)was added to 15 ml of 0.2M NaOH and allowed to dissolve overnight toform a homogeneous solution. Five (5) ml of the hyaluronic acid that wasneutralized with 1M HCl solution was mixed with 50 mg of difunctionallyactivated S-PEG in 0.5 ml of PBS using syringe-to-syringe mixing.

The resulting material was extruded from the syringe into a petri dishand incubated at 37° C. After 16 hours, the material had formed acrosslinked gel.

Hyaluronic acid without S-PEG was used as a control in this experiment.After 16-hour incubation, the control was still liquid and runny.

Example 2

Forty (40) mg of difunctionally activated S-PEG was mixed with 145 ul of1M HCl. After thorough mixing, the acidified S-PEG solution was drawninto a syringe.

A 6.6% (w/v) solution of deacetylated hyaluronic acid was prepared bymixing hyaluronic acid with 0.2M NaOH. The deacetylated hyaluronic acidsolution (pH 13) was also transferred to a syringe.

The two syringes were then connected with a 3-way stopcock and thecontents mixed using syringe-to-syringe mixing. Mixing the acidifiedS-PEG with the deacetylated hyaluronic acid caused the pH of thesolution to neutralize and the crosslinking reaction to occur.

After mixing for 60-70 passes, the material was transferred to onesyringe. The stopcock and the second (empty) syringe were removed. Thematerial was now ready for injection and in situ crosslinking.

Example 3

One (1) milliliter of 35 mg/ml collagen in solution (pH 2) is mixed with1 ml of a 2% (w/v) acidified solution of difunctionally activated S-PEG.The S-PEG- collagen solution is immediately mixed with 2 ml of a 10mg/ml solution of deacetylated hyaluronic acid (pH 13), neutralizing thepH of the mixture and causing the difunctionally activated S-PEG tocovalently bond with both the collagen and the hyaluronic acid.

Example 4

One (1) milliliter of 35 mg/ml Zyderm® I Collagen and 1 ml of a 10 mg/mlsolution of hyaluronic acid are mixed together at pH 10. Thecollagen-hyaluronic acid solution is then mixed with 2 ml of a 2% (w/v)solution of difunctionally activated S-PEG in 0.1M HCl (pH 1), causingthe solution to neutralize and crosslinking to occur between PEG,collagen, and hyaluronic acid.

Example 5

One (1) milliliter of 35 mg/ml Zyderm® I Collagen (pH 7) is mixed with 1ml of a 10 mg/ml solution of deacetylated hyaluronic acid in 0.2M NaOH(pH 13). Two (2) milliliters of a 4% (w/v) solution of acidifieddifunctionally activated S-PEG is immediately added to neutralize the pHand effect crosslinking between the three components.

Example 6

One (1) milliliter of Zyderm® I Collagen and 1 ml of a 10 mg/mo solutionof deacetylated hyaluronic acid are each adjusted to pH 9, then mixedtogether. The collagen-hyaluronic acid solution is then adjusted toapproximately pH 7 by adding 0.1M HCl, causing the hyaluronic acid andcollagen to form a weak gel due to ionic interaction. Subsequentaddition of difunctionally activated S-PEG results in covalentcrosslinking, producing a strong gel.

Example 7 (Coating of Implants)

Prepare a hyaluronic acid--S-PEG reaction mixture as described inExample 1. Dip a titanium implant into the reaction mixture immediatelyafter crosslinking is initiated. Allow the implant coating to finishcrosslinking, and dry overnight.

The present invention is shown and described herein at what isconsidered to be the most practical and preferred embodiments. It isrecognized, however, that departures may be made therefrom which arewithin the scope of the invention and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

What is claimed:
 1. A biocompatible, biologically inert conjugatecomprising a difunctionally activated hydrophilic synthetic polymerchemically conjugated to both collagen, or a derivative thereof, and toa glycosaminoglycan, or a derivative thereof.
 2. The conjugate of claim1, wherein the conjugate has the following structural formula:

    GAG-HN-OC-(CH.sub.2).sub.n -Z-PEG-Z-(CH.sub.2).sub.n -CO-NH-COL

wherein n is an integer ranging from 0 to about 4 and GAG is aglycosaminoglycan or a derivative thereof; COL is collagen, or aderivative thereof; and Z is O or O--C═O.
 3. The conjugate of claim 2,wherein the glycosaminoglycan is selected from the group consisting ofhyaluronic acid, the chondroitin sulfates, heparin, keratan sulfate,keratosulfate, chitin, chitosan 1, chitosan 2, and derivatives thereof,and mixtures of these glycosaminoglycans or their derivatives.
 4. Theconjugate of claim 2, wherein the collagen is selected from the groupconsisting of fibrillar collagen or nonfibrillar collagen.
 5. Theconjugate of claim 2, in a form selected from the group consisting of amembrane, bead, sponge, tube, sheet, and formed implant.
 6. Theconjugate of claim 2, in the form of a hydrogel composition having amoisture content in the range of from about 5% to about 95%.
 7. Theconjugate of claim 5, in the form of a formed implant for use in therepair, augmentation, or replacement of a body part selected from thegroup consisting of a heart valve, patella, ear, nose, and cheekbone. 8.The conjugate of claim 2, in the form of a bodily fluid replacement forjoint fluid or vitreous humor.